Overexpression of certain growth factor receptors such as epidermal growth factor receptor (EGFR) as well as the closely related c-erbB2, also known as HER2, are observed in many human cancers including bladder cancer (1), colon carcinoma (2) and lung cancer (3). In breast cancer, high levels of EGFR (4) and erbB2 (5) correlate strongly with poor prognosis. More importantly, these receptors can intensify the transforming signal in a synergistic manner through their ability to form both homo- and heterodimers (6). It has now been shown that by activation of a certain growth factor, the epidermal growth factor receptor (EGFR) induces expression of DNA repair enzymes including ERCC1 and XRCC1, two DNA repair enzymes that are involved in nucleotide excision (NER) and base-excision repair (BER) of alkylated adducts. Tumors overexpressing EGFR are resistant to apoptosis, leading to reduced sensitivity to anti-tumor drugs.
Agents capable of blocking disordered growth signaling, mediated by the tyrosine kinase (TK) activity of these receptors, are now used or are in clinical trials against breast cancer (7, 8). Herceptin (trastuzamad), a humanized antibody against erbB2, showed a 22% response rate as a single agent in metastatic breast cancer (7). ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase (TK), is now in phase III clinical trials (8).
Agents targeting EGFR and erbB2 present two major advantages. Firstly, they induce selective antitumor activities and secondly, they exhibit a good toxicity profile with only mild side effects. However, where they cannot induce apoptosis, they are cytostatic agents capable of inducing reversible anti-tumor effects.
The anilinoquinazolines are considered the most potent class of EGFR TK inhibitors acting through competitive inhibition of ATP in the TK domain (9, 10, 11). More precisely, anilinoquinazolines inhibit EGFR-related signal transduction by competitive binding in the ATP site. Moreover, a significant number of structure-activity-relationship (SAR) studies on 4-anilinoquinazolines and pyrido[d]pyrimidines as EGFR TK inhibitors have accumulated to suggest that the compounds bind to the ATP site of EGFR. Recently, irreversible inhibitors of EGFR and erbB2 have been developed based on the quinazoline class, by appending an acryloyl group to the 6-position of the anilinoquinazolines as exemplified by PD168393 (Scheme 1).

These inhibitors, containing a Michael acceptor at the 6-position, showed greater potency than their reversible predecessors (12, 13, 14). Their mechanism of action is based on the specific alkylation of Cys-773 of EGFR and erbB2, leading to a covalent inactivation and irreversible inhibition of these receptors. Molecular modeling suggests that the N-1 atom accepts an H-bond from Met-769 and N-3 from the side chain of Thr-766 on strand 5 deep in the binding cleft. The anilino moiety binds in an adjacent hydrophobic pocket.
The binding mode of quinazoline in the ATP site of EGFR has recently been confirmed by X-ray crystallography. These models suggest that the only positions on the inhibitors where substituents can be altered without affecting binding affinity are the 6- and 7-positions which are located at the entrance of binding cleft. Indeed, a variety of compounds with bulky side chains on the 6- and 7-positions have been synthesized and found to retain significant binding affinity for the EGFR ATP binding site.
Fry et al. (13) (Park-Davis Pharmaceutical Research, Division of Warner-Lambert) demonstrated that an acrylamide moiety (Michael acceptor), appended to the 6-position of a quinazoline, adopts the appropriate orientation in order to react with the nucleophilic thiol atom of Cys-773. The distance between these groups was measured as being not greater than 2.8 Å. In contrast, the 7-position is oriented at a distance greater than 7 Å, and the 7-acrylamide substituted analogues alkylate EGFR at a considerably slower rate than those in which it is appended to the 6-position.
More recently, Discafini et al, (15) and Tsou et al. (16) (Wyeth-Ayerst Research, A Division of American Home Products), developed novel compounds bearing different types of Michael acceptors such as butynamides or cyclic α,β-unsaturated ketones. The choice of Michael acceptors is inspired by their mild alkylating activity, a property that is considered critical for specific alkylation of the cystein residue of EGFR to occur.
In order to further potentiate the action of EGFR TK inhibitors, studies have been designed to combine them with classical cytotoxic drugs (8, 17, 18). Within the same line of idea, Jean-Claude et al. have recently developed a novel tumor targeting strategy, termed “combi-targeting”, that seeks to develop novel drugs designated as “combi-molecules” capable of blocking growth factor-mediated signaling while inducing cytotoxic DNA damage. The “combi-targeting” strategy consists of combining a cytotoxic DNA damaging function with an EGFR inhibitory property into single molecule i.e. “combi-molecule”. These “combi-molecules” are designed to release to the two moieties (i.e. the cytotoxic DNA damaging function and the EGFR inhibitory function) upon hydrolysis. Furthermore, Jean-Claude et al. have recently demonstrated that following cell penetration, these combi-molecules require hydrolytic scission to generate the cytotoxic function (19, 20, 21). The combi-targeting strategy is based on the premise that blocking EGFR-mediated signaling via a kinase inhibitory component and damaging DNA by an alkylating function will induce a tandem block of EGFR activation and that of DNA repair enzymes required to rescue the cells. This in turn is expected to lead to synergistic killing of EGFR-overexpressing cells.
The first prototypes of combi-molecules contained a quinazoline head and a triazene (ZRBA1) or a nitrosourea tail (FD137) (Scheme 2). These combi-molecules were designed not only to bind to the receptor on their own, but also to degrade to an aminoquinazoline capable of further blocking EGFR TK and an alkylating species. While the binary potency of these compounds has now been well demonstrated, they were all designed to release a stoichiometric amount to the free inhibitor in addition to the release of a stoichiometric amount of an alkylating species.

There thus remains a need for the development of a new prototype of combi-molecule having increased tumor selectivity and improved efficacy in refractory tumors. Moreover specifically, there remains a need to develop a new prototype type of combi-molecule that does not require hydrolysis to generate the mixed EGFR and DNA targeting properties. There also remains a need to develop a process for the synthesis of novel potent inhibitors of epidermal growth factor receptor having fluorescent properties. Finally, there remains a need to develop biomarkers having high selectivity for EGFR.
The present invention seeks to meet these and other needs.
The present invention refers to a number of documents, the content of which is herein incorporated by reference in their entirety.